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AIM : Weill�/Marchesani syndrome (WMS) is a raresystemic disorder with both autosomal recessive anddominant inheritances. Accumulation of reactiveoxygen species such as O2

+�, H2O2 and OH+ causeslipid peroxidation (LPO), whereas antioxidant en-zymes (superoxide dismutase (SOD), glutathioneperoxidase (GSHPx)) mediate defence against oxida-tive stress. Excess tumour necrosis factor (TNF)-a andNO+ react with O2

+� and cause further antioxidantdepletion with an increase in mutation frequency byH2O2. This study investigated the levels of SOD,GSHPx , catalase (CAT), TNF-a, NO+ and LPO inpatients with WMS.Methods : A group of 10 WMS patients (four males, sixfemales; age, 26.59/19.0 years) and 10 age-matchedand sex-matched controls (five males, five females;age, 27.39/18.2 years) were included. Serum TNF-alevels were determined by a spectrophotometertechnique using immulite chemiluminescent immu-nometric assay. Malondialdehyde (MDA) was deter-mined in plasma; CAT in red blood cells (RBCs), andSOD and GSHPx in both plasma and RBCs. Totalserum NO+ levels were evaluated by Griess reaction.Results : Mean levels of TNF-a (8.39/0.6 pg/ml) inWMS patients were significantly (p B/0.001) higherthan controls (4.39/0.2 pg/ml). Plasma MDA levels inpatients and controls were 5.49/0.8 and 1.89/0.6mmol/l, respectively, and the difference was signifi-cant (p�/0.0002). SOD and GSHPx activities weresignificantly lower in both RBCs and plasma of WMSthan in controls (RBC-SOD, 3981.99/626.6 versus5261.69/523.0 U/g haemoglobin (Hb), p�/0.0005;plasma-SOD, 529.49/49.3 versus 713.49/55.7 U/g pro-tein, p�/0.0002; RBC-GSHPx , 682.79/42.0 versus756.59/47.6 U/g Hb, p�/0.0011; plasma-GSHPx ,107.39/15.0 versus 131.49/19.7 U/g protein,p�/0.0113). In addition, serum NO+ (NO�

2 �/NO�3 )

levels were also significantly (p�/0.0002) increasedin WMS patients (54.49/5.7 versus 26.99/6.7 mmol/l).RBC-CAT levels were similar between groups (125.69/

21.3 versus 131.09/21.5 k/g Hb, p�/0.8798).Conclusions : The elevated LPO, TNF-a and NO+ withdecreased antioxidant enzyme activities indicatedimpaired antioxidative defence mechanisms with anoxidative injury and cell toxicity in WMS patients.The use of multiple antioxidants and free radicalscavengers might be helpful in this genetic disorder.

Key words: Antioxidant enzymes, Lipid peroxidation,Nitric oxide, Tumour necrosis factor a, Weill�/Marchesanisyndrome

Mediators of Inflammation, 13(3), 165�/170 (Month 2004)

Tumour necrosis factor a, lipidperoxidation and NO+ areincreased and associated withdecreased free-radical scavengingenzymes in patients with Weill�/

Marchesani syndrome

Cem Evereklioglu1,CA, Yusuf Turkoz2,

Mustafa Calis3, Fuat Duygulu4 and

Aysun B. Karabulut2

1Department of Ophthalmology, 3Department ofPhysical Medicine and Rehabilitation, and4Department of Orthopaedics and Traumatology,Erciyes University Medical Faculty, Kayseri, Turkey;2Department of Biochemistry, Inonu UniversityMedical Faculty, Turgut Ozal Medical Centre, Malatya,Turkey

CACorresponding Author:Tel:�/90 352 233 15 65Fax: �/90 352 235 83 65E-mail: [email protected]

Introduction

In normal cells, a balance exists between oxidativedamage and antioxidant defence. Although tumournecrosis factor (TNF)-a and highly potent oxidantmolecules such as superoxide anion (O2

+�), hydro-gen peroxide (H2O2) and hydroxyl radical (OH+) areproduced by the immune system, excessive produc-tion of oxygen-derived free radical creates a condi-

tion known as oxidative stress, participating in

various diseases and syndromes.1�7

Antioxidant defences in humans protect directly

and indirectly the host against the damaging influ-

ence of such cytokines and oxidants stated earlier.1

These enzymes include superoxide dismutase (SOD,

EC1.15.1.1), glutathione peroxidase (GSHPx ,

EC1.11.1.9) and catalase (CAT, EC1.11.1.6). First,

SOD catalyses the dismutation of O2+� to H2O2.

Research Communication

ISSN 0962-9351 print/ISSN 1466-1861 online/04/30165-06 – 2004 Taylor & Francis LtdDOI: 10.1080/09511920410001713547

165

GSHPx and CAT then independently convert H2O2 toH2O.8 If H2O2 is produced in excess, it reacts withO2

+� and produces OH+, which is one of the mostactive reactive oxygen species (ROS). The ROSinteracts with nucleic acids, proteins and lipids, thusenhancing TNF production, DNA mutations, cellulardysfunction and death.1,2

Nitric oxide (NO+) is one of the most abundantfree radicals in the body.4,5 Excess NO+ productioncauses respiratory enzyme inhibition of mitochon-dria, resulting in the cessation of DNA replication.The rapid reaction of NO+ with other radicals such asO2

+� to form the powerful oxidant is the generalmechanism that greatly enhances the toxicity of NO+.Although O2

+� is scavenged by SOD, NO+ is the onlyknown molecule that can be produced high enoughto out-compete SOD for O2

+�.9

Weill�/Marchesani syndrome (WMS) was first re-ported by Weill10 and Marchesani.11 It is a congenitalconnective tissue disease characterised by shortstature, brachydactyly, microspherophakia, glau-coma, and ectopia lentis. Although we have pre-viously reported a large family with autosomaldominant transmission,12 recessive mode of inheri-tance has also been described.13 However, the exactaetiology of this unique disorder is unknown and theautosomal dominant form has been linked to muta-tion within the fibrillin-1 gene on chromosome15q21.1, whereas autosomal recessive WMS mappedto chromosome 19p13.3-p13.2.13,14

In the present study, red blood cell (RBC) activitiesof SOD, GSHPx and CAT, and plasma activities ofSOD and GSHPx were measured for the first time in10 patients with autosomal dominant WMS andcompared with age-matched and sex-matchedhealthy controls. Plasma TNF-a, malondialdehyde(MDA) and NO levels were also investigated.

Patients and methods

Ten patients with WMS (four males, six females) fromtwo families showing dominant transmission and 10age-matched and sex-matched healthy control sub-jects (five males, five females) were included in thisstudy. Systemic and detailed ocular examinationswere performed in all subjects in both groups. Allpatients with WMS had the classical systemic andocular findings including brachymorphy (n�/10 , allbelow or equal to the third percentile), brachydactyly(n�/10 , stubby fingers and/or clumsy feet), spher-ophakia or microspherophakia (n�/9), high refrac-tive error (n�/6) and lens subluxation (n�/6) withiridophacodonesis. Three patients demonstrated bi-lateral but asymmetric glaucomatous optic nervedamage with increased intraocular pressure. In addi-tion, vitreous liquefaction (n�/9), strabismus (n�/2),

microcornea (n�/1), scleral staphyloma (n�/1) andlimited mobility of joints (n�/5) were also present.All control subjects had normal systemic and ocularfindings.

Blood collection

The laboratory personnel were maintained masked tothe clinical diagnosis and group of the subjects,matching each blood sample by letter coding, andso were the clinicians to subsequent levels until theend of the study. After informed consent wasobtained from all patients and controls, antecubitalwhole-blood samples were drawn from a peripheralvein using a 25-gauge needle, avoiding haemolysis,into evacuated heparinised plain tubes in the morn-ing hours (08:30�/10:30) after an overnight fast and 15min of supine rest. None of the patients and controlsreceived any topical or systemic medication at least 4weeks prior to blood collection. The samples werecentrifuged at 3000 g for 10 min at 48C, and theharvested plasma was collected and kept at�/508Cuntil the time of analysis.

The erythrocytes were subsequently washed twicewith two volumes of 0.9% sodium chloride solutionto remove the plasma remnants. Following this, theerythrocytes were haemolysed with two-fold vo-lumes of ice-cold distilled water. After centrifugation(5000 g , 10 min, 48C) the supernatant was subdividedand transferred into polyethylene tubes and frozen at�/708C to be used in the assay of FRSEs inerythrocytes (SOD, GSHPx and catalase). The plasmawas used to measure the end product of lipidperoxidation (LPO) (MDA), TNF-a, total nitrite/ni-trate levels (NO�

2 �/NO�3 ) as an indicator of NO

levels, and antioxidant enzymes (SOD, GSHPx , CAT).The haemoglobin (Hb) content of whole blood wasalso measured, and enzyme activity was determinedper mg Hb.

Cytokine analysis

Cytokine analysis was performed according to theImmulite† (Diagnostic Products Corporation, LosAngeles, CA, USA) chemiluminescent enzyme immu-nometric assay. The technique is based on a solid-phase (bead) two-site assay. The solid phase, apolystyrene bead, is coated with a monoclonalspecific antibody. After the incubation for 30�/60min at 378C, unbound conjugate is then removed by acentrifugal wash (three times), after which a chemi-luminescent substrate (a phosphate ester of adaman-tyl dioxetane) is added, and the test unit is incubatedfor a further 10 min. The Immulite† system auto-matically handles sample and reagent additions, theincubation and separation step, and measurement ofthe photon output via the temperature-controlledluminometer. It calculates test results for control and

C. Evereklioglu et al.

166 Mediators of Inflammation � Vol 13 � 2004

patient samples from the observed signal, using astored master curve, and generates a printed report.15

The values of inter-assay imprecision were similarto those from the intra-assay study with coefficient ofvariation (CV) ranging from 2% to 11.5%. The CV forthe measured cytokine was around 5%. The linearityis satisfactory, with a regression coefficient higherthan 0.99 (r2).

There is an excellent practicality of the system andgood stability of the calibration curve (15 days). It is agood reliable method yielding a good precision alongwith a satisfactory detection limit.16 The antibodyused in the Immulite† for TNF-a is highly specific foreach cytokine and chemokine, with no cross-reactiv-ity to other cytokines that may be present in theserum samples.

A master curve is constructed by the manufacturerusing a material calibrated against the NationalInstitute for Biological Standards and Control. Anadjustment of the calibration slope is made by theuser by measuring two-serum matrix vials (low andhigh) designated as ‘adjusters’. It should be men-tioned that between-run and within-run imprecisiondata were similar, which is very important for statmeasurement.17

Plasma MDA analysis

LPO is frequently investigated in biomedical researchand the assays for thiobarbituric acid-reactive sub-stances (TBARS) are much more widely used thanany other index of LPO in biological samples.18

Thiobarbituric acid (TBA) reacts with LPO aldehydes,such as MDA. Therefore, assessment of TBARS is auseful indice of oxidative deterioration and LPOdetermination in body fluids.

Plasma MDA level was measured according to themethod described by Wasowicz et al .19 The principalof the method is based on the coupling of TBARSwith TBA. All measurements (standards and samples)were performed at the upper n -butanol phase of thereaction mixture. In brief, 50 ml of plasma or anadequate volume of MDA working standard solutionwas introduced into 10 ml glass tubes containing 1 mlof distilled water. After addition of 1 ml of thesolution containing 29 mmol/l TBARS in acetic acidand mixing, the samples were placed in a water bathand heated for 1 h at 95�/1008C. After the sampleswere cooled, 25 ml of 5 mol/l HCI was added, and thereaction mixture was extracted by agitation for 5 minwith 3.5 ml of n -butanol. Butanol phase wasseparated by centrifugation at 1500 g for 10 min.The butanol extract was measured with a spectro-fluorometer (F-4010 fluorescence spectrophotometer;Hitachi, Tokyo, Japan) at wavelengths of 525 nm forexcitation. The calibration curve was prepared withMDA standards of 0�/10 mmol/l. Intra-assay and inter-

assay CVs were 3.5% and 6%, respectively. Resultswere expressed as nanomoles per millilitre.

Plasma total nitrite/nitrate (NO�2 �/NO�

3 )analysis

Because total nitrite is the stable end product of NO+

metabolism, NO+ synthesis was determined as totalnitrite using a spectrophotometric assay based on theGriess reaction as described previously.20�24 In short,samples (250 ml) were incubated at room temperaturewith 250 ml of substrate buffer (0.1 mol/l of imidazole,210 mmol/l of NADPH, 3.8 mmol/l of flavine adeninedinucleotide; pH 7.6) in the presence of nitratereductase (Aspergillus niger ; Sigma, St. Louis, MO,USA) for 45 min to convert nitrate (NO�

3 ) to nitrite(NO�

2 ). Excess reduced NADPH, which interfereswith the chemical detection of nitrite, was oxidisedby continuation of the incubation of 5 mg (1 ml) ofLDH (Sigma), 0.2 mmol/l (120 ml) of pyruvate (Sigma)and 79 ml of water. Total nitrite was then analysed byreacting the samples with Griess reagent (1% sulfa-nilamide, 0.1% naphthalene�/ethylene diamine dihy-drocholoride in 5% H3PO4 spectroquant; Merck,Darmstadt, Germany). Reacted samples were treatedwith 500 ml of trichloroacetic acid (20%), centrifugedfor 15 min at 8000 g and the absorbance at 548 nmwas compared with that of NaNO2 standard (0�/100mmol/l). Total nitrite/nitrate levels were expressed asmicromoles per litre.

RBCs and plasma-SOD analysis

SOD activity in plasma and supernatant was mea-sured according to the method of Sun et al .25 bydetermining the inhibition of nitroblue tetrazolium(NBT) reduction with xanthine�/xanthine oxidaseused as an O2

+� generator. Activity was assessed inthe ethanol phase of the lysate after 1.0 ml of ethanol/chloroform mixture (5/3, v/v) was added to the samevolume of the hemolysate and centrifuged. One unitof SOD is defined as the amount of protein or Hb thatinhibits the rate of NBT reduction by 50%. Resultswere defined as units per gram of protein orhaemoglobin (U/g protein or U/g Hb).

RBCs and plasma GSHPx analysis

GSHPx activity in plasma and supernatant wasmeasured according to the method of Paglia andValentine.26 Enzyme activity was determined from theoxidation of reduced NADPH in the presence ofH2O2 used as substrate. The decrease in concentra-tion of NADPH was monitored and recorded at 340nm in a mixture containing reducted glutathione andglutathione reductase (pH 7.8, 258C). Enzyme unitswere defined as the number of micromoles ofNADPH oxidised per minute. Results were defined

TNF-a, LPO oxidative stress in Weill�/Marchesani syndrome

Mediators of Inflammation � Vol 13 � 2004 167

as international units per gram of protein or of Hb (U/g protein or U/g Hb).

RBC-CAT analysis

CAT activity in supernatant was determined accord-ing to the method of Aebi27 by monitoring the initialrate of disappearance of H2O2 (initial concentration,10 mM) at 240 nm (e�/0.041/mmol�/1/cm) in acuvette containing 10.5 mM H2O2 in 1 ml of 50 mMphosphate buffer (pH 7, 258C), in a spectrophot-ometer. Results were reported as the constant rate persecond per gram of Hb (K/g Hb). Protein concentra-tions in plasma samples were measured according toLowry et al .28

Statistical analysis

The Mann�/Whitney U -test was used for the statistics,and the results expressed as mean9/standard devia-tion. The two-tailed statistical significance betweentwo means was considered p B/0.05. Statistical ana-lysis was performed with Statistical Package for theSocial Sciences for Windows (release 7.5; SPSS/PC�/

Inc., Chicago, IL, USA).

Results

The mean age between patients (26.59/19.0 years;range, 6�/68 years) and controls (27.39/18.2 years;range, 8�/65 years) was comparable (p �/0.05). Themean value of TNF-a (8.39/0.6 pg/ml) was signifi-cantly (p B/0.001) higher in WMS than control sub-jects (4.39/0.2 pg/ml). RBC activities of SOD(3981.99/626.6 U/g Hb) and GSHPx (682.79/42.0U/g Hb) were significantly lower in WMS patientswhen compared with controls (5261.69/523.0 U/gHb, p�/0.0005 and 756.59/47.6 U/g Hb, p�/0.0011,respectively) (Table 1). The RBC-CAT activity ofpatients with WMS (125.69/21.3 k/g Hb) was com-parable with controls (131.09/21.5 k/g Hb,p�/0.8798). The plasma levels of SOD (529.49/49.3

U/g protein) and GSHPx (107.39/15.0 U/g protein)were also significantly lower in WMS patients thanthose in controls (713.49/55.7 U/g protein, p�/0.0002and 131.49/19.7 U/g protein, p�/0.0113, respec-tively). Plasma MDA levels in WMS patients andcontrols were 5.49/0.8 mmol/l and 1.89/0.6 mmol/l,respectively, and the difference was significant (p�/

0.0002). Plasma NO concentrations in WMS patients(54.49/5.7 mmol/l) were also significantly (p�/

0.0002) higher than in healthy control subjects(26.99/6.7 mmol/l).

Discussion

WMS is a very rare systemic connective tissuedisorder with systemic and ocular findings.12 Brachy-morphy, brachydactyly and, significantly, myopiawere noted among parents and other relatives,suggesting that the gene might be dominant with aseverely reduced penetrance or a wide range ofexpressivity.13,29 Alternatively, the syndrome may beconsidered incompletely recessive with partial ex-pression in the heterozygote.30 Although GEMSSsyndrome (glaucoma�/ectopia�/microspherophakia�/

stiff joints�/short stature) has been suggested as anacceptable name for the autosomal dominant form,31

the relationship between the dominant and recessiveWMS is not known. Therefore, early differentialdiagnosis is mandatory, especially in subtle cases,for prognostic, therapeutic and genetic reasons(genetic counselling) in order to make a nosologicdiagnosis and to prevent potentially life-threateningsystemic complications.

Increased oxidative damage with decreased SODactivity has been suggested in the pathogenesis ofDown syndrome as well as in the ageing process.32

Electron microscopic study of the microspherophakiclens obtained from a patient with WMS clearlydemonstrated epithelial cell degeneration, markedliquefaction and destruction of the subcapsularcortical fibres.33 Such degeneration and necrosis ofthe cells are caused by various factors including

Table 1. Plasma LPO, NO+ and antioxidant enzyme activities in plasma and erythrocytes of patients with WMS and controls

Weill�/Marchesani syndrome(n�/10; four male, six female)

Healthy controls(n�/10; five male, five female)

P*

Age (years) 26.59/19.0 27.39/18.2 0.8205Plasma-MDA (mmol/l) 5.49/0.8 1.89/0.6 0.0002RBCs-SOD (U/g Hb) 3981.99/626.6 5261.69/523.0 0.0005Plasma-SOD (U/g protein) 529.49/49.3 713.49/55.7 0.0002RBC-GSHPx (U/g Hb) 682.79/42.0 756.59/47.6 0.0011Plasma-GSHPx (U/g protein) 107.39/15.0 131.49/19.7 0.0113RBC-CAT (k/g Hb) 125.69/21.3 131.09/21.5 0.8798TNF-a (pg/ml) 8.39/0.6 4.39/0.2 B/0.001Plasma NO (mmol/l) 54.49/5.7 26.99/6.7 0.0002

Data presented as mean9/standard deviation.* Mann �/Whitney U-test.



ageing, inflammation and trauma.34 Destruction ofthe cortical fibres is a common histological changeseen in lenses with senile cortical cataracts.35 There-fore, premature ageing process may participate inWMS.

In the present study, we have studied plasmaLPO and TNF-a levels with the antioxidant systemSOD, CAT and GSHPx in both RBCs and plasma ofpatients with WMS. Both plasma and RBCs activitiesof SOD and GSHPx were found to be decreasedwhereas RBC-CAT activities did not changed.In addition, plasma MDA, TNF-a and NO+ levelswere also significantly increased when comparedwith controls. Because free radicals play a sign-ificant role in the multifactorial syndrome, whichconstitutes systemic and ocular manifestations,our results suggest that both RBCs and serum ofWMS patients have an unbalanced antioxidantsystem with increased LPO and TNF-a, thus participating in the manifestations of this uniquedisease.

Free radicals are mainly derived from a univalentsequential reduction of molecular oxygen. Mitochon-dria are the main location of intracellular production,which may also result from auto-oxidation of smallmolecules or function of some enzymes. The free-radical theory of cell dysfunction is basically foundon three main observations: (1) free radicals areextremely reactive species; (2) the production of oxyfree radicals, mainly O2

+�, is a constant phenomenonin the organism with beneficial (defence againstmicrobial aggression by phagocytes) and damagingeffects at the cellular and molecular levels; and (3)natural defence or control mechanisms occur byenzymatic antioxidants. Therefore, an imbalancebetween production and control mechanisms issupposed to result in the continuous or progressiveaccumulation of deleterious changes throughout thecells and tissues, thus generating intense functionaldisorder at each level of organisation of ultrastructures, cells and organs.36

The cell defence system against free-radical-in-duced toxic LPO (MDA), referred to TBARS, consistof antioxidative free-radical scavenging moleculessuch as SOD, GSHPx and CAT.37 These enzymesblock the initiation of free-radical chain reactions.Many syndromes such as Hutchinson�/Gilford, Wer-ner’s and Down’s syndromes are genetically con-trolled. Because genetic mutations result indistributed activities of the antioxidative enzymessuch as SOD, GSHPx and CAT, the genomic and free-radical theories are closely linked as the altered SOD/(GSHPx plus CAT) ratio may affect gene expressionby altering the binding and/or availability of tran-scription factors to DNA.37,38 Indeed, this studydemonstrated that there was a significant increasein LPO characterised by elevated plasma MDA levelsin patients with WMS when compared with healthy

controls. It is well known that increased production

of free radicals as a result of decreased antioxidant

enzymes SOD and GSHPx can lead to the formation

of LPO.7 In addition, both erythrocyte and plasma

SOD levels as well as GSHPx activities were sig-

nificantly decreased when compared with controls. If

SOD levels decrease concomitantly with GSHPx ,

then the first (O2+�) and second step (H2O2) inter-

mediate radicals accumulate. These oxygen free

radicals could undergo the Fenton’s reaction, gen-

erating hydroxyl radicals, which may lead to LPO in

cells. Therefore, the increased plasma LPO levels in

this syndrome may affect the susceptible target

tissues more seriously.It should be noted that the interaction between

NO+ and O2+�, producing a cytotoxic oxidant,

peroxynitrite, has recently received a great deal of

attention.39 Indeed, decreased SOD and GSHPx

activities paired with increased NO+ react with

O2+� and cause further antioxidant depletion and,

therefore, oxidative damage.There is a debate whether a relation exists between

the activation of the inflammatory mediator cascade

and changes of the organism’s antioxidative system.

Because free radicals are among the key mediators of

a TNF-a-induced killing event, TNF-a-induced apop-

tosis may be mediated by free radicals and LPO in

WMS.40 In other words, this study suggests that

increased TNF-a may be a key contributor to the

cascade that establishes the tissue injury process of

oxidant-derived cell injury. Thus, immune dysfunc-

tion and increased oxidation and LPO seem to

participate during the course of this disorder.In conclusion, although much than more is known

about the systemic and ocular manifestations of WMS

with attempts to define still the gene, the exact

aetiology is still unclear and, to our knowledge, there

is no molecular study performed on WMS patients.

Many other biochemical changes within the body,

especially in molecular basis, cannot be ruled out.

Increased LPO and NO+ levels with decreased

antioxidant enzyme activities found in the present

study may suggest that NO+, O2+� and H2O2 are

generated in excess resulting in oxidative stress in

WMS. These changes may affect cellular macromole-

cules as well as nucleic acids, leading to DNA

mutation and the cessation of DNA replication. Our

results are consistent with the notion that ROS may

contribute to the process of cellular dysfunction in

mammalians. Therefore, further studies in a larger

group of patients are needed in this respect to

confirm this interpretation and hypothesis.

ACKNOWLEDGEMENT. The authors have no financial or proprietaryinterest in any instrument or products used in this study.

TNF-a, LPO oxidative stress in Weill�/Marchesani syndrome

Mediators of Inflammation � Vol 13 � 2004 169

References

1. Grimble RF. Nutritional antioxidants and the modulation of inflamma-tion: theory and practice. New Horiz 1994; 2: 175�/185.

2. Pastor MC, Sierra C, Dolade M, et al . Antioxidant enzymes and fatty acidstatus in erythrocytes of Down’s syndrome patients. Clin Chem 1998;44: 924�/929.

3. Ben�/Menachem E, Kyllerman M, Marklund S. Superoxide dismutaseand glutathione peroxidase function in progressive myoclonus epilep-sies. Epilepsy Res 2000; 40: 33�/39.

4. Evereklioglu C, Er H, Turkoz Y, Cekmen M. Serum levels of TNF-a, sIL-2R, IL-6, and IL-8 are increased and associated with elevated lipidperoxidation in patients with Behcet’s disease. Mediat Inflamm 2002;11: 87�/93.

5. Evereklioglu C, Er H, Doganay S, et al . Nitric oxide and lipidperoxidation are increased and associated with decreased antioxidantenzymes in patients with age-related macular degeneration. DocOphthalmol 2003; 106: 129�/136.

6. Erkilic K, Evereklioglu C, Cekmen M, Ozkiris A, Duygulu F, Dogan H.Adenosine deaminase enzyme activity is increased and negativelycorrelates with catalase, superoxide dismutase and glutathione perox-idase in patients with Behcet’s disease: original contributions/clinicaland laboratory investigations. Mediat Inflamm 2003; 12: 107�/116.

7. Beckman KB, Ames BN. The free radical theory of aging matures.Physiol Rev 1998; 78: 547�/581.

8. Chance B, Sies H, Boveris A. Hydroperoxide metabolism in mammalianorgans. Physiol Rev 1979; 59: 527�/605.

9. Sozmen EY, Kerry Z, Uysal F, et al . Antioxidant enzyme activities andtotal nitrite/nitrate levels in the collar model. Effect of nicardipine. ClinChem Lab Med 2000; 38: 21�/25.

10. Weill G. Ectopia des crystallins et malformations generales. Ann Oculist1932; 169: 21�/44.

11. Marchesani O. Brachydactylie und angeborene Kugellinse als System-erk-rankung. Klin Monatsbl Augenheilkd 1939; 103: 392�/406.

12. Evereklioglu C, Hepsen IF, Er H. Weill�/Marchesani syndrome in threegenerations. Eye 1999; 13: 773�/777.

13. Faivre L, Megarbane A, Alswaid A, et al . Homozygosity mapping of aWeill�/Marchesani syndrome locus to chromosome 19p13.3-p13.2. HumGenet 2002; 110: 366�/370.

14. Wirtz MK, Samples JR, Kramer PL, et al . Weill�/Marchesani syndrome*/

possible linkage of the autosomal dominant form to 15q21.1. Am J MedGenet 1996; 65: 68�/75.

15. Babson AL. The IMMULITE Automated Immunuassay System. J Im-munoassay 1991; 14: 83�/88.

16. Berthier F, Lambert C, Genin C, Bienvenu J. Evaluation of an automatedimmunoassay method for cytokine measurement using the ImmuliteImmunoassay system. Clin Chem Lab Med 1999; 37: 593�/599.

17. Doganay S, Evereklioglu C, Er H, et al . Comparison of serum NO, TNF-a, IL-1b, sIL-2R, IL-6 and IL �/8 levels with grades of retinopathy inpatients with diabetes mellitus. Eye 2002; 16: 163�/170.

18. Lapenna D, Ciofani G, Pierdomenico SD, Giamberardino M, CuccurulloF. Reactions conditions affecting the relationship between thiobarbituricacid reactivity and lipid peroxides in human plasma. Free Radic BiolMed 2001; 31: 331�/335.

19. Wasowicz W, Neve J, Perets A. Optimized steps in flourometricdetermination of thiobarbituric acid-reactive substances in serum:Importance of extraction pH and influence of sample preservation andstorage. Clin Chim 1993; 39: 2522�/2526.

20. Granger DL, Taintor RR, Boockvar KS, Hibbs JB Jr. Measurement ofnitrite and nitrate in biological samples using nitrate reductase andGriess reaction. Methods Enzymol 1996; 268: 142�/151.

21. Er H, Doganay S, Turkoz Y, et al . The levels of cytokines and nitric oxidein rabbit vitreous humor after retinal laser photocoagulation. Ophthal-mic Surg Lasers 2000; 31: 479 �/483.

22. Doganay S, Evereklioglu C, Turkoz Y, Er H. Decreased nitric oxideproduction in primary open �/angle glaucoma. Eur J Ophthalmol 2002;12: 44�/48.

23. Evereklioglu C, Turkoz Y, Er H, Inaloz HS, Ozbek E, Cekmen M.Increased serum nitric oxide production in patients with Behcet’sdisease: is it a new activity marker? J Am Acad Dermatol 2002; 46:50�/54.

24. Evereklioglu C, Cekmen M, Ozkiris A, Karabas L, Calis M. Thepathophysiological significance of red blood cell nitric oxide concentra-tions in inflammatory Behcet’s disease. Mediat Inflamm 2003; 12: 255�/

256.25. Sun Y, Oberley LW, Li Y. A simple method for clinical assay of

superoxide dismutase. Clin Chem 1988; 34: 479�/500.26. Paglia DE, Valentine WN. Studies on the quantitative and qualitative

characterisation of erythrocyte glutathione peroxidase. J Lab Clin Med1967; 70: 158�/169.

27. Aebi H. Catalase in vitro. Methods Enzymol 1984; 105: 121�/126.28. Lowry OH, Rosenbrough NJ, Farr AL. Protein measurement with the

Folin phenol reagent. J Biol Chem 1951; 193: 265�/275.29. Kloepfer HW, Rosenthal JW. Possible genetic carriers in the

spherophakia�/brachymorphy syndrome. Am J Hum Genet 1955; 7:398�/425.

30. Jensen AD, Cross HE, Paton D. Ocular complication in the Weill�/

Marchesani syndrome. Am J Ophthalmol 1974; 77: 261�/269.31. Verloes A, Hermia JP, Galand A, Koulischer L, Dodinval P. Glaucoma �/

Lens Ectopia�/Microspherophakia �/Stiffness�/Shortness (GEMSS) syn-drome. Am J Med Genet 1992; 44: 48�/51.

32. Gerli G, Zenoni L, Locatelli GF, et al . Erythrocyte antioxidant system inDown syndrome. Am J Med Genet Suppl 1990; 7: 272�/273.

33. Fujiwara H, Takigawa Y, Ueno S, Okuda K. Histology of the lens in theWeill �/Marchesani syndrome. Br J Ophthalmol 1990; 74: 631�/634.

34. Klinthworth GK, Garner A. The cause, types and morphology ofcataracts. In: Garner A, Klinthworth G, eds. Pathology of Ocular Disease.New York: Dekker, 1982: 1223�/1272.

35. Evereklioglu C, Alasehirli B, Sari I, Cengiz B, Bagci C. Effect of nicotineexposure during gestation on neonatal rat crystalline lenses. Eye 2004;18: 67�/73.

36. Crastes de Paulet A. Free radicals and ageing. Ann Biol Clin 1990; 48:323�/330.

37. Guemouri L, Artur Y, Herbeth B, Jeandel C, Cuny G, Siest G. Biologicalvariability of superoxide dismutase, glutathione peroxidase, and catalasein blood. Clin Chem 1991; 37: 1932�/1937.

38. Knight JA. The biochemistry of ageing. Adv Clin Chem 2000; 35: 1�/62.39. Muijers RB, Folkerts G, Henricks PA, Sadeghi-Hashjin G, Nijkamp FP.

Peroxynitrite: a two-faced metabolite of nitric oxide. Life Sci 1997; 60:1833�/1845.

40. Lee DH, Macintyre JP, Taylor GR, et al . Tepoxalin enhances the activityof an antioxidant, pyrrolidine dithiocarbamate, in attenuating tumornecrosis factor alpha-induced apoptosis in WEHI 164 cells. J PharmacolExp Ther 1999; 289: 1465�/1471.

Received 19 January 2004Accepted 18 March 2004



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